Reverse transcription-PCR Results Research Articles

Comparative evaluation of RT-PCR and antigen-based rapid diagnostic tests (Ag-RDTs) for SARS-CoV-2 detection: performance, variant specificity, and clinical implications.

The COVID-19 pandemic has highlighted the critical need for accurate and efficient diagnostic tools for detecting severe acute respiratory coronavirus 2 (SARS-CoV-2) infections. This study presents a comparison of two diagnostic tests: RT-PCR and antigen detection rapid diagnostic tests (Ag-RDTs). This study focused on their performance, variant specificity, and their clinical implications. A simultaneous testing of 268 samples was carried out for SARS-CoV-2 using RT-PCR and Ag-RDTs [flourescence immunoassay (FIA) and lateral flow immunoassay (LFIA)]. Viral load was quantified, and variant identification was performed using a PCR-based assay. The prevalence was found to be 30.2% using reverse transcription PCR (RT-PCR), 26.5% using FIA, and 25% using LFIA. When comparing the FIA and LFIA, the overall diagnostic performance was found to be 80.25% vs 76.54%, 96.79% vs 97.33%, 91.55% vs 90.51%, and 91.88% vs 92.56% for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. Both Ag-RDTs showed a strong agreement with RT-PCR (κ = 0.78-0.80). The overall accuracies of the FIA and LFIA were 92.41% and 92.13%, respectively. The FIA showed higher sensitivity (73.68%) and PPV (92.08%) than the LFIA (65.79% and 90.56%, respectively) in asymptomatic patients. At low Ct values (<25), both Ag-RDTs had 100% sensitivity, but the sensitivity reduced to 31.82% for FIA and 27.27% for LFIA at Ct values > 30. The diagnostic sensitivity of FIA compared to LFIA for detecting the Alpha variant was 78.85% vs. 69.23% and 72.22% vs. 83.33% for the Delta variant. Both Ag-RDTs had 100% sensitivity for detecting Omicron. Both Ag-RDTs performed well in patients with high viral loads and Omicron variant infections compared to those infected with Alpha and Delta variants. This study confirms the comparable performance of RT-PCR and Ag-RDTs, specifically FIA and LFIA, for SARS-CoV-2 detection. The FIA showed higher sensitivity and PPV in asymptomatic cases, while both Ag-RDTs exhibited strong agreement with RT-PCR results. Notably, Ag-RDTs, particularly FIA, proved effective in detecting the Omicron variant and cases with high viral loads, highlighting their potential clinical utility in managing the COVID-19 pandemic.IMPORTANCEThis study is of utmost importance in providing effective responses to manage the COVID-19 pandemic. It rigorously compares the diagnostic accuracy, variant specificity, and practical considerations of reverse transcription PCR (RT-PCR) and antigen detection rapid diagnostic tests (Ag-RDTs) for severe acute respiratory coronavirus 2 (SARS-CoV-2), answering critical questions. The results of this study will help healthcare professionals choose the appropriate testing methods, allocate resources effectively, and enhance public health strategies. Given the evolution of the virus, understanding the performance of these diagnostic tools is crucial to adapting to emerging variants. Additionally, the study provides insights into logistical challenges and accessibility issues, which will contribute to refining testing workflows, particularly in resource-limited settings. Ultimately, the study's impact extends to global healthcare, providing valuable information for policymakers, clinicians, and public health officials as they work together for mitigating the impact of the pandemic.

Cloning and Analysis of Expression of Genes Related to Carotenoid Metabolism in Different Fruit Color Mutants of Pepper (Capsicum annuum L.).

The formation of fruit color in pepper is closely related to the processes of carotenoid metabolism. In this study, red wild-type pepper XHB, SP01, PC01 and their corresponding mutants H0809 (orange), SP02 (yellow), and PC02 (orange) were used as research materials. The Ggps, Psy, Lcyb, Crtz, Zep, and Ccs genes involved in carotenoid biosynthesis were cloned, and bioinformatics and expression analyses were carried out. The results showed that the full lengths of the six genes were 1110 bp, 2844 bp, 1497 bp, 2025 bp, 510 bp, and 1497 bp, and they encoded 369, 419, 498, 315, 169, and 498 amino acids, respectively. Except for the full-length Ccs gene, which could not be amplified in the yellow mutant SP02 and the orange mutant PC02, the complete full-length sequences of the other genes could be amplified in different materials, indicating that the formation of fruit color in the SP02 and PC02 mutants could be closely related to the deletion or mutation of the Ccs gene. The analytical results of real-time quantitative reverse transcription PCR (qRT-PCR) showed that the Ggps, Psy, Lcyb, Crtz, and Zep genes were expressed at different developmental stages of three pairs of mature-fruit-colored materials, but their patterns of expression were not consistent. The orange mutant H0809 could be amplified to the full Ccs gene sequence, but its expression was maintained at a lower level. It showed a significant difference in expression compared with the wild-type XHB, indicating that the formation of orange mutant H0809 fruit color could be closely related to the different regulatory pattern of Ccs expression. The results provide a theoretical basis for in-depth understanding of the molecular regulatory mechanism of the formation of color in pepper fruit.

Identification and function of the Pax gene Bmgsb in the silk gland of Bombyx mori.

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.

Roles of Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway in Cd-induced testicular injury in rats and the protective effect of quercetin

Cadmium (Cd) exposure causes oxidative damage to mitochondria, which would adversely affect rat testicular tissue. Quercetin (Que) is a natural antioxidant with anti-inflammatory, antioxidant and anti-apoptotic effects. However, the mechanism by which Que inhibits Cd-induced apoptosis of testicular cells remains unclear. The purpose of this study was to investigate the role of mitochondrial apoptosis pathway (Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway) in inhibiting Cd-induced apoptosis of testicular cells by Que. We used SD rats to simulate Cd chloride exposure by treating all sides of the rats with CdCl2 and/or Que. The levels of GSH and MDA in rat testis were detected using reagent kits. The effects of CdCl2 and/or Que on tissue damage, apoptosis, and gene and protein expression of the Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway in rat testis were examined by HE, TUNEL, RNA extraction and reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blot (Wb). The results show that Cd significantly increased the contents of GSH and MDA in rat testis (P < 0.01); conversely, Que significantly reduced the contents of GSH and MDA (P < 0.01). Cd inflicted damage to testicular tissue, and Que addition significantly reduced the damage. Cd increased the number of apoptosis of testicle cells, and Que inhibited testicle-cell apoptosis. In addition, the results of reverse transcription PCR and Wb assays confirmed that, as expected, Cd increased the expression levels of Cyt-c, Caspase-9, Caspase-3, and Bax mRNAs as well as proteins. And at the same time decreased the expression of the anti-apoptotic factor Bcl-2 in the cells. Surprisingly, these effects were reversed when Que was added. Therefore, Que can play an antioxidant and anti-apoptotic role in reducing the testicular tissue damage caused by Cd exposure. This provides a conceptual basis for the later development and utilization of Que as well as the prevention and treatment of tissue damage caused by Cd exposure.

Efflux pump inhibitory potential of indole derivatives as an arsenal against norA over-expressing Staphylococcus aureus.

NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay.

Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.

Genetic Variability and Molecular Evolution of Tomato Mosaic Virus Populations in Three Northern China Provinces.

RNA viruses tend to mutate during transmission and host infection, which is critical to viral adaptation and evolution. Tomato mosaic virus (ToMV) is a member of the genus Tobamovirus (family Virgaviridae) and an economically important virus with detrimental effects on tomatoes worldwide. Although the ToMV gene sequences have been completed in China, their genetic diversity and population structure remain unclear. We collected 425 tomato samples from tomato-growing areas in three northern Chinese provinces 2016. Reverse transcription PCR results showed that the average incidence of the virus in the field samples was 67.15%, and ToMV was detected in all test areas. The analysis of ToMV single nucleotide polymorphisms in China showed that ToMV was evolutionarily conserved, and the variation in the whole genome was uneven. Pairwise identity analysis showed significant variability in genome sequences among ToMV strains with genomic nucleotide identities of 73.2-99.6%. The ToMV population in the northern Chinese provinces had purification and selection functions, which were beneficial in the evolution of the ToMV population. Although there has been some distribution of ToMV strains in China, the virus was generally stabilized as a uniform strain under the pressure of purification selection. Our findings show how to monitor the prevalent strains of ToMV and their virulence in China and provide useful information for its prevention and control.

Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai.

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1-28 aa), a transforming growth factor-β (TGF-β) propeptide (38-272 aa), and a mature TGF-β peptide (314-416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.

Causes of fetal death in the Flemish cattle herd in Brazil.

Flemish cattle in Brazil are on the brink of extinction and are found only in one herd in Lages, Santa Catarina State. This study aimed to uncover the reasons for the recurring abortions in the Flemish cattle herd. Seventeen Flemish fetuses underwent postmortem examinations, with samples collected for histopathology and microbiology culture tests, polymerase chain reaction (PCR) test for Neospora caninum, and reverse transcription-PCR (RT-PCR) test for bovine viral diarrhea virus (BVDV) from 2015 to 2020. Of the 17 fetuses, N. caninum was the most common diagnosis and was found in 88% (15/17). One fetus (5.8%) had a coinfection with N. caninum and Citrobacter amalonaticus, leading to fibrinonecrotic pericarditis. All fetuses tested negative for BVDV by RT-PCR. Of the 107 dams tested by indirect immunofluorescence assay, 26 (25.2%) were anti-N. caninum seropositive, with 17 (65.4%) aborting and 5 (19.2%) having estrus repetition. Reverse transcription-PCR results showed that 9 (8.4%) of the serum samples collected from dams tested positive, which tested follow-up test 3 months later, indicating a BVDV transient infection. The factors that contributed to neosporosis included dogs' access to pastures and improper disposal of fetal remains, which made it easier for dogs to consume them. This study warns the occurrence of N. caninum as a cause of reproductive disorders that can lead to abortion in the studied Flemish cattle herd.

The TLR4/NF-κB signaling pathway-mediated type 2 skewing of T helper cell in cough variant asthma was counteracted by ethanol extract of Anacyclus pyrethrum root

Type 2 T helper (Th2) cells-mediated immune response plays a pivotal role in the pathogenesis of cough variant asthma (CVA), and this study aims to determine the effect and mechanism of ethanol extract of Anacyclus pyrethrum root (EEAP) on regulating Th2 response in CVA. Peripheral blood mononuclear cells (PBMCs) collected from patients with CVA, and naive CD4+T cells induced by Th2-polarizing medium were administrated with EEAP. Interestingly, through conducting flow cytometry and enzyme linked immunosorbent assay method, we found that EEAP significantly alleviated Th2 skewing and increased Th1 response in these two kinds of cells. Results of western blot assay and quantitative reverse transcription PCR displayed that EEAP suppressed the expression of TLR4, total NF-κB p65, nuclear NF-κB p65 and the downstream genes. Subsequently, we proved that TLR4 antagonist E5564 played a similar improvement role to EEAP in Th1/Th2 imbalance, while combination of TLR4 agonist LPS and EEAP abolished the inhibitory effect of EEAP on Th2 polarization in Th2-induced CD4+T cells. Finally, CVA models induced by ovalbumin and capsaicin were established in cavies, and data showed that EEAP also improved Th1/Th2 imbalance in CVA in vivo, manifested in the increase of IL4+CD4+T cell ratio, Th2 cytokines (IL-4, IL-5, IL-6 and IL-13) and the decrease of Th1 cytokines (IL-2 and IFN-γ). Co-treatment of LPS and EEAP counteracted the inhibition of EEAP on Th2 response in CVA model cavies. Moreover, we found that EEAP mitigated airway inflammation and hyper-responsiveness in vivo, which was abolished by the combined application of LPS. In a word, EEAP restores Th1/Th2 balance in CVA through restraining the TLR4/NF-кB signaling pathway. This study may contribute to the clinical application of EEAP in CVA-related disease.

Transcriptome analysis reveals the crucial function of hyperoside in inhibiting anthocyanin accumulation in grape (Vitis vinifera L.) fruits by inducing VvMYB62.

The formation of color in plants is significantly dependent on anthocyaninpigments. Grape species vary in color due to the differences in anthocyanin accumulation. It is widely recognized that both biotic and abiotic conditions may have an impact on anthocyanin synthesis in plants. The underlying molecular mechanisms by which external application of hyperoside impacts anthocyanin formation in grapes, however, have received little attention. In the current study,the transcriptome of Gemstone seedless grape was examined using high-throughput RNA sequencing at various developmental stages reply to both control and hyperoside treatments. The results of this study suggested that the major genes controlling anthocyanin accumulation in response to the externalinjection of hyperoside could be VvMYB62, VvPAL, VvCHS, and VvF3'5'H.Quantitative reverse transcription PCR (RT-qPCR) results were used to confirm the changes in the expression levels of the genes encoding the anthocyanin biosynthesis pathway under the control and hyperoside treatments. Using a transient transformation system, it was discovered that VvMYB62 was shown to regulate the anthocyanin accumulation at both the transcriptional and posttranslational levels and could be influenced by the external administration of hyperoside. In grape embryogenic calli, hyperoside could specifically suppress theexpression of VvMYB62 and anthocyanin accumulation. In this instance, the VvMYB62 characterisation brought attention to the significance of exogenous hyperoside-induced anthocyanin accumulation. Therefore, the results demonstrated that VvMYB62 could be hindered in the process of grape during anthocyanin accumulation caused by hyperoside. These findings offer excellent candidate genes in the future breeding of novel grape varieties in addition to serving as a crucial reference for understanding the underlying molecular processes of hyperoside suppression of anthocyanin formation in plants.

Development of the differential detection reverse transcription loop-mediated isothermal amplification method for Barley yellow mosaic virus or Japanese soil-borne wheat mosaic virus from barley.

A differential detection reverse transcription loop-mediated isothermal amplification (DD-RT-LAMP) method was developed to detect either Barley yellow mosaic virus (BaYMV) or Japanese soil-borne wheat mosaic virus (JSBWMV) simultaneously. Both primer sets, which recognized either BaYMV or JSBWMV genomic RNA, amplified DNA more efficiently at 65°C using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak annealing temperatures of BaYMV and JSBWMV amplification products using specific primer sets were 86.9°C-87.7°C and 84.5°C-85.0°C, respectively, and were clearly distinguishable during an annealing step following the isothermal amplification, monitored using a fluorescence detection device. In the field samples of barley (Hordeum vulgare L.) tested, BaYMV or JSBWMV were detected by DD-RT-LAMP, and the detection results of DD-RT-LAMP were correspondent with the results of reverse transcription-PCR.

Evaluation of microRNA let-7b-3p expression levels in methamphetamine abuse

In this study, we aimed to identify a microRNA expression signature that could be used to distinguish methamphetamine from control samples. We also utilized the existing bioinformatics tools in order to predict the candidate microRNAs that could play potential key roles in regulating drug addiction-related genes. Methamphetamine samples from 21 ventral tegmental area and 21 nucleus accumbens samples and their control regions were obtained from the Council of Forensic Medicine (Istanbul). Quantitative analysis of let-7b-3p was studied using quantitative reverse transcription PCR. Statistical analysis was carried out using Student's t-test. The receiver operating characteristic curves were plotted with Statistical Package for the Social Sciences (SPSS 20.0). Our quantitative reverse transcription PCR results revealed that let-7b-3p was significantly overexpressed in brain tissues of the methamphetamine-user group. Let-7b-3p had significant power to discriminate methamphetamine from control samples in the ventral tegmental area (AUC; 0.922) and nucleus accumbens (AUC; 0.899) regions. We have shown for the first time in the literature the differential expression of let-7b-3p in samples from methamphetamine-addicted individuals. We suggest that let-7b-3p could be a powerful marker for the diagnosis of methamphetamine addiction. Our results showed that differentially expressed let-7b-3p in methamphetamine users could be used as a diagnostic and therapeutic marker.

Genomic footprints of Kam Sweet Rice domestication indicate possible migration routes of the Dong people in China and provide resources for future rice breeding

The Dong people are one of China’s 55 recognized ethnic minorities, but there has been a long-standing debate about their origins. In this study, we performed whole-genome resequencing of Kam Sweet Rice (KSR), a valuable, rare, and ancient rice landrace unique to the Dong people. Through comparative genomic analyses of KSR and other rice landraces from south of the Yangtze River Basin in China, we provide evidence that the ancestors of the Dong people likely originated from the southeast coast of China at least 1000 years ago. Alien introgression and admixture in KSR demonstrated multiple migration events in the history of the Dong people. Genomic footprints of domestication demonstrated characteristics of KSR that arose from artificial selection and geographical adaptation by the Dong people. The key genes GS3, Hd1, and DPS1 (related to agronomic traits) and LTG1 and MYBS3 (related to cold tolerance) were identified as domestication targets, reflecting crop improvement and changes in the geographical environment of the Dong people during migration. A genome-wide association study revealed a candidate yield-associated gene, Os01g0923300, a specific haplotype in KSR that is important for regulating grain number per panicle. RNA-sequencing and quantitative reverse transcription–PCR results showed that this gene was more highly expressed in KSR than in ancestral populations, indicating that it may have great value in increasing yield potential in other rice accessions. In summary, our work develops a novel approach for studying human civilization and migration patterns and provides valuable genomic datasets and resources for future breeding of high-yield and climate-resilient rice varieties.

Modulation of the p38 MAPK Pathway by Anisomycin Promotes Ferroptosis of Hepatocellular Carcinoma through Phosphorylation of H3S10.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. Ferroptosis is emerging as an effective target for tumor treatment as it has been shown to potentiate cell death in some malignancies. However, it remains unclear whether histone phosphorylation events, an epigenetic mechanism that regulates transcriptional expression, are involved in ferroptosis. Our study found that supplementation with anisomycin, an agonist of p38 mitogen-activated protein kinase (MAPK), induced ferroptosis in HCC cells, and the phosphorylation of histone H3 on serine 10 (p-H3S10) was participated in anisomycin-induced ferroptosis. To investigate the anticancer effects of anisomycin-activated p38 MAPK in HCC, we analyzed cell viability, colony formation, cell death, and cell migration in Hep3B and HCCLM3 cells. The results showed that anisomycin could significantly suppress HCC cell colony formation and migration and induce HCC cell death. The hallmarks of ferroptosis, such as abnormal accumulation of iron and elevated levels of lipid peroxidation and malondialdehyde, were detected to confirm the ability of anisomycin to promote ferroptosis. Furthermore, coincubation with SB203580, an inhibitor of activated p38 MAPK, partially rescued anisomycin-induced ferroptosis. And the levels of p-p38 MAPK and p-H3S10 were successively increased by anisomycin treatment. The relationship between p-H3S10 and ferroptosis was revealed by ChIP sequencing. The reverse transcription PCR and immunofluorescence results showed that NCOA4 was upregulated both in mRNA and protein levels after anisomycin treatment. And by C11-BODIPY staining, we found that anisomycin-induced lipid reactive oxygen species was reduced after NCOA4 knockdown. In conclusion, the anisomycin-activated p38 MAPK promoted ferroptosis of HCC cells through H3S10 phosphorylation.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes

To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes. The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag. The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05). We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Diagnostic Performance Assessment of Saliva RT-PCR and Nasopharyngeal Antigen for the Detection of SARS-CoV-2 in Peru.

ABSTRACTWidely available and reliable testing for SARS-CoV-2 is essential for the public health response to the COVID-19 pandemic. We estimated the diagnostic performance of reverse transcription PCR (RT-PCR) performed on saliva and the SD Biosensor STANDARD Q antigen test performed on nasopharyngeal swab compared to the reference standard, nasopharyngeal swab (NP) RT-PCR. We enrolled participants living and/or seeking care in health facilities in North Lima, Peru from November 2020 to January 2021. Consenting participants underwent same-day RT-PCR on both saliva and nasopharyngeal swab specimens, antigen testing on a nasopharyngeal swab specimen, pulse oximetry, and standardized symptom assessment. We calculated sensitivity, specificity, and predictive values for the nasopharyngeal antigen and saliva RT-PCR compared to nasopharyngeal RT-PCR. Of 896 participants analyzed, 567 (63.3%) had acute signs/symptoms of COVID-19. The overall sensitivity and specificity of saliva RT-PCR were 85.8% and 98.1%, respectively. Among participants with and without acute signs/symptoms of COVID-19, saliva sensitivity was 87.3% and 37.5%, respectively. Saliva sensitivity was 97.4% and 56.0% among participants with cycle threshold (CT) values of ≤30 and >30 on nasopharyngeal RT-PCR, respectively. The overall sensitivity and specificity of nasopharyngeal antigen were 73.2% and 99.4%, respectively. The sensitivity of the nasopharyngeal antigen test was 75.1% and 12.5% among participants with and without acute signs/symptoms of COVID-19, and 91.2% and 26.7% among participants with CT values of ≤30 and >30 on nasopharyngeal RT-PCR, respectively. Saliva RT-PCR achieved the WHO-recommended threshold of >80% for sensitivity for the detection of SARS-CoV-2, while the SD Biosensor nasopharyngeal antigen test did not.IMPORTANCE In this diagnostic validation study of 896 participants in Peru, saliva reverse transcription PCR (RT-PCR) had >80% sensitivity for the detection of SARS-CoV-2 among all-comers and symptomatic individuals, while the SD Biosensor STANDARD Q antigen test performed on nasopharyngeal swab had <80% sensitivity, except for participants whose same-day nasopharyngeal RT-PCR results showed cycle threshold values of <30, consistent with a high viral load in the nasopharynx. The specificity was high for both tests. Our results demonstrate that saliva sampling could serve as an alternative noninvasive technique for RT-PCR diagnosis of SARS-CoV-2. The role of nasopharyngeal antigen testing is more limited; when community transmission is low, it may be used for mass screenings among asymptomatic individuals with high testing frequency. Among symptomatic individuals, the nasopharyngeal antigen test may be relied upon for 4 to 8 days after symptom onset, or in those likely to have high viral load, whereupon it showed >80% sensitivity.

Fifteen-Month Follow-Up of Anti-Spike Receptor-Binding Domain SARS-CoV-2 Antibodies among Healthcare Workers in Boston, MA.

Boston Medical Center (BMC) is a safety net hospital in Boston, and from the initial wave of COVID-19 there has been an overwhelming concern about the exposure of healthcare workers (HCWs) to SARS-CoV-2. We conceived a study to follow a cohort of BMC HCWs, beginning in July 2020 and continuing for 15 months, collecting survey data and serum samples at approximately 3-month intervals. Serum samples were analyzed using the Abbott Architect i2000 for SARS-CoV-2 antibodies (anti-spike1-Receptor Binding Domain IgG and anti-nucleoprotein IgG). Positive anti-n IgG results were used, in addition to reverse transcription-PCR results, for identifying cases of infection. History of COVID-19 and vaccination status were confirmed, where possible, using electronic medical records. Participants were grouped according to vaccination and infection status in September 2021 for analysis of anti-s IgG trends. A majority of HCWs remain well above the positivity threshold for anti-spike IgG antibodies for up to 11 months post-vaccination and 15 months post-infection, regardless of combinations and permutations of vaccination and infection. Those with COVID-19 infection before vaccination had significantly higher median serum antibody concentrations in comparison to HCWs with no prior infection at each follow-up time point. These findings further support what is known regarding the decline in serum antibody concentrations following natural infection and vaccination, adding knowledge of serum antibody levels for up to 15 months post- infection and 11 months post-vaccination.

Juvenile hormone acts on male accessory gland function via regulating l-asparaginase expression and triacylglycerol mobilization in Aedes aegypti.

Hormones control the reproductive development of Aedes aegypti mosquitoes. The adult male reproductive process and mating behavior require adequate nutrients and energy. Understanding the molecular mechanism linking hormones, energy metabolism, and reproduction in male mosquitoes is important. In this study, we found that the size of the male accessory gland, an essential part of the male reproductive system, gradually increased after eclosion. However, it was significantly reduced in male mosquitoes deficient in methoprene-tolerant (Met), the receptor of juvenile hormone. Likewise, egg hatchability of females that mated with Met-depleted males showed the same downward trend. The mRNA level of the gene encoding accessory gland protein, l-asparaginase (ASNase), was reduced in Met dsRNA-treated males. Electrophoretic mobility shift assay and quantitative reverse transcription-PCR results revealed that Met was capable of binding directly to the promoter of ASNase and activated its transcription. RNA interference of ASNase in males resulted in the reduction of egg hatchability of the females with which they mated. These results showed that Met influenced the fecundity of male mosquitoes by directly upregulating the expression of the ASNase gene. Moreover, the levels of triacylglycerol and the sizes of lipid droplets were decreased by 72-78h after eclosion in the fat body cells, whereas both of them increased in Met-depleted male mosquitoes, indicating that Met knockdown reduced lipid catabolism. These data demonstrate that Met might influence the egg hatchability of females by regulating lipid metabolism and the development of the male accessory gland in male mosquitoes.

Fusobacterium necrophorum Promotes Apoptosis and Inflammatory Cytokine Production Through the Activation of NF-κB and Death Receptor Signaling Pathways.

Fusobacterium necrophorum can cause liver abscess, foot rot in ruminants, and Lemire syndrome in humans, Also, its virulence factors can induce the apoptosis of macrophages and neutrophils. However, the detailed mechanism has not been fully clarified. This study investigated the mechanisms of apoptosis and inflammatory factor production in F. necrophorum–induced neutrophils and macrophages (RAW246.7). After infection of macrophages with F. necrophorum, 5-ethynyl-2’-deoxyuridine labeling assays indicated that F. necrophorum inhibited macrophage proliferation in a time- and dose-dependent manner. Hoechst staining and DNA ladder assays showed significant condensation of the nucleus and fragmentation of genomic DNA in F. necrophorum–infected macrophages, Annexin V (FITC) and propidium iodide (PI) assay confirmed the emergence of apoptosis in the macrophages and sheep neutrophils with F. necrophorum compared with the control. The group with significant apoptosis was subjected to RNA sequencing (RNA-Seq), and the sequencing results revealed 2581 up– and 2907 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed genes showed that F. necrophorum drove apoptosis and production of inflammatory factors by activating genes related to the Nuclear Factor-κB (NF-κB) and death receptor pathways. Meanwhile, quantitative reverse transcription PCR and Western blot validation results were consistent with the results of transcriptome sequencing analysis. In conclusion, F. necrophorum induced apoptosis and production of pro-inflammatory factors through the NF-κB and death receptor signaling pathway, providing a theoretical basis for further mechanistic studies on the prevention and control of F. necrophorum disease treatment.